TMC GENES We have generated mice with knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their hearing and balance function. Mice that are homozygous for the Tmc1 knockout allele are deaf. Mice that are homozygous for the Tmc2 knockout allele have normal hearing and balance. Mice that are homozygous for knockout alleles of both genes are deaf and have abnormal balance function. These results indicate that both Tmc1 and Tmc2 are required for normal balance function, whereas only Tmc1 is required for hearing. We are currently working to identify the location and function of TMC1 and TMC2 proteins in sensory hair cells of the hearing and balance organs. We collaborated with Dr. Guy Van Camp and Rick Friedman to demonstrate the genetic origin of a TMC1 mutation causing progressive deafness in a large family. This mutation was identical to a TMC1 mutation that we had identified in an unrelated family. The results indicate that the mutations arose independently and this nucleotide position appears to be a hot spot for mutations and encodes an amino acid that is critical for the function of the TMC1 protein. We have studied the location and structure of mouse TMC1 protein expressed in tissue culture cells. TMC1 is located in the membranes of the endoplasmic reticulum in this system. We used a differential detergent treatment protocol to determine the transmembrane topology of this protein: i.e. what segments of the protein are located in the cytosolic compartment and what segments are located in the lumen of the endoplasmic reticulum. The results demonstrate that the amino- and carboxy-terminal ends of the protein are cytosolic and that these termini are separated by six membrane-spanning domains. This structural organization resembles that of other proteins known to be important for transporting ions across plasma membranes and suggests that TMC proteins also act as transmembrane pumps, channels, transporters, or receptors. We used a yeast two-hybrid screen to isolate genes encoding proteins that potentially interact with TMC1. We narrowed the list to a few candidate genes. We are using a combination of approaches to determine which interactions occur in hair cells. We generated knockout mice for Tmc6 and Tmc8 to better understand the function(s) of Tmc genes and proteins. Mutations in human TMC6 or TMC8 genes cause epidermodysplasia verruciformis, a recessive disease resulting in chronic cutaneous HPV infections (papillomas or warts) with increased susceptibility to non-melanoma skin cancers. We have done extensive RNA expression analyses to show that Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues and lung and skin, and primarily during development. The homozygous knockout mice have no obvious phenotypic abnormalities, so we are collaborating with Dr. Paul Lambert to determine if these mice have alterations in their susceptibility or response to papillomavirus infection. TWIRLER MOUSE We have identified a candidate mutation and generated a knock-in mouse line with this mutation to confirm its pathogenicity. The resulting line has all of the phenotypic features of Twirler, thus proving the pathogenic role of the candidate mutation. We are currently studying the effects of this mutation on expression of the adjacent gene and characterizing how changes in the expression of this gene lead to inner ear malformations and cleft palate and lip. ENLARGED VESTIBULAR AQUEDUCTS (EVA) We are ascertaining families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. We are using those families in a genetic linkage-based strategy to identify other genetic causes of EVA. We are evaluating several candidate regions of potential linkage. We studied the SLC26A4 protein function associated with mutant alleles from our EVA patients to determine if the level of residual function might be associated with the severity of the thyroid and hearing phenotypes. There are also a number of SLC26A4 variants found as the only variant allele in EVA patients and we sought to determine whether these are benign coincidental variants or whether they are pathogenic mutations. By assessing how often these mutations were found in patients with Pendred syndrome and/or in association with a second mutant allele, we devised a way to predict which variants are likely to be pathogenic and which are not. We studied the segregation (inheritance) of EVA in families of patients with only one or no detectable SLC26A4 mutations. The frequency of detection of EVA in the siblings of EVA patients was the same in patients with one or two mutations, indicating that we are probably simply missing the second mutation in patients with only one detectable mutation. IN contrast, the frequency was much lower if the patient has no mutations, suggesting that EVA is not being inherited as a simple single-gene recessive trait in those patients and families. We performed comprehensive assessments of the thyroid gland in patients with EVA. We concluded that ultrasound evaluation of thyroid gland volume is recommended for the initial assessment and follow-up surveillance in patients with EVA. We performed a correlative study of SLC26A4 genotype, cochlear radiologic structure and hearing loss in a large cohort of EVA patients. We showed that the number of mutant alleles of SLC26A4, but not the presence of cochlear structural anomalies, is correlated with hearing loss severity in ears with EVA. We generated a doxycycline-inducible Slc26a4-expression mouse line. This transgenic mouse line allows us to manipulate Slc26a4 expression (on an Slc26a4-knockout background) by the administration of doxycycline in drinking water. We have defined a time window during which Slc26a4 is required for auditory development and function. We can manipulate doxycycline administration to generate mice in which there is significant residual hearing and isolated EVA, a phenotype which models human EVA more closely than the existing knockout mouse. We are currently working to identify the structural and functional changes within the cochleae of these mice to characterize the pathogenesis of hearing loss. COLLABORATIVE PROJECTS We collaborated with Dr. Thomas Friedman of the NIDCD to identify the structure, location and function of the human deafness protein TRIOBP in neurosensory hair cells of the mouse inner ear. We collaborated with Drs. Hong-Joon Park and Un-Kyung Kim to identify the mutation of the DFNA5 gene in a Korean family with autosomal dominant progresive hearing loss. We showed that this mutation has also been identified in other east Asian families with hearing loss, and that the mutation identified in these families probably arose from an ancient ancestral founder.